A large increase in concentrations of cytosolic Ca2 ([Ca2+]c) generally causes release of prolactin, but there are two conditions in which TRH causes a marked rise in [Ca2+]c but prolactin release is inhibited: one is after pretreatment with dopamine and the other is after frequent pulses of TRH. Because TRH generates a large spike of [Ca2+]c in these two cases, the inhibition of prolactin release must occur at a step subsequent to the use in [Ca2+]c. The goal is to understand the mechanisms of inhibition by determining what processes in the cell are no longer able to respond to increases in [Ca2+]c. The specific aims are: 1. To determine if the activation of the protein kinase C system by TRH still occurs when prolactin release is inhibited by dopamine pretreatment or by frequent TRH pulses. 1a) We will determine what isozymes of protein kinase C are present in normal lactotrophs and if they are still translocated by TRH when prolactin release is inhibited. 1b) We will determine if TRH is still capable of phosphorylating a well-characterized substrate of protein kinase C when prolactin release is inhibited. 2. To investigate whether calmodulin-dependent kinases can be activated by TRH when prolactin release is inhibited. 2a) We will determine whether elongation factor II, the substrate for Ca2+/calmodulin kinase III, can still by phosphorylated by TRH when prolactin release is inhibited. 2b) We will investigate whether TRH causes changes in the phosphorylation state of Ca2+/calmodulin kinase II and if the changes still occur when release is inhibited. In these first two aims, if we find effects, we will characterize them. 3. To isolate and characterize proteins whose phosphorylation is stimulated by TRH when prolactin release occurs but not when prolactin release is inhibited. We will determine what kinases can phosphorylate these proteins and why phosphorylation is inhibited when prolactin release is inhibited. This aim will extend knowledge of mechanisms of inhibition beyond the characterized systems described in the first two aims. The information gained from these experiments will increase our understanding of desensitization that occurs beyond the level of second messengers.